Pipelines, Robots, Crystals and Biology: What Use High Throughput Solving Structures of Challenging Targets?

Abstract

With recent advances in the technology and software underlying crystallographic structure solution, demands on both output and functional significance of X-ray structures are soaring. To achieve the required speed and quality also with ever larger and more difficult targets, combining HTP screening methods (robotics based or not) adopted from structural genomics initiatives with thorough expertise and dedicated characterization effort for each individual target is almost a must. I present concepts, practical considerations, and experiences on implementing an HTP technology platform for structural and functional studies on complexes, membrane proteins and other challenging targets. Emphasis lies on the environment of small academic groups engaged exclusively in hypothesis driven projects focused on specific biological systems. Suitability of given HTP protocols for particular target classes, benchmarking and quality control for procedures, and project management issues at the interface between extensive, broad parameter screening and intensive individual target work required by non-SG amenable targets are discussed.

Molecular dynamics simulations of potassium channels

“…Such an expansion in the number of membrane proteins is due to the fact that technological advances have allowed structural biology to reach a position where the elucidation of the three-dimensional structure of relevant proteins on a large scale appears possible. At the same time high-throughput approaches are emerging [8][9][10] to characterize membrane proteins and analyze their topology, issues of significant interest for drug discovery. Unfortunately, the expression and crystallization of integral membrane proteins, such as ion channels, is still fraught with difficulties.…”

Getting a Grip on Complexes

“…Consequently, structural genomics intensively stimulated and fostered the implementation of automation and high-throughput approaches, which now result also in considerable benefit for classical, hypothesis driven structural molecular biology. Many laboratories are now in the process of integrating high-throughput approaches at varying levels in their research [49]. …”

“…Among them, recombination based cloning systems are most widely utilized in high-throughput experiments. Although the systems used currently are robust and can be automated, they are often not sufficiently flexible when variations of expression elements such as purification tags, promoter/terminator combinations, protease cleavage sites and others need to be introduced or modified [49]. …”

Expression and purification of an adenylation domain from a eukaryotic nonribosomal peptide synthetase: Using structural genomics tools for a challenging target

“…One potential strategy to tackle this technical challenge is to make use of the highthroughput cloning and expression screening systems [11,12] that have been developed for structural genomics programs over the past decade. In the context of structural genomics, the high-throughput tools are used to screen large numbers of genes for the production of soluble proteins that can then be screened for crystal growth and structure determination.…”